Two cytoplasmic 5'-nucleotidases of Bacillus subtilis K

• Published in: Journal of biochemistry (Tokyo) Vol. 85; no. 4; pp. 1083 - 1089
• Main Authors: OZAKI, Hachiro, SHIIO, Isamu
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 01.04.1979
• Subjects: Bacillus subtilis - enzymology; Kinetics; Magnesium - pharmacology; Nucleotidases - isolation & purification; Nucleotidases - metabolism; Subcellular Fractions - enzymology; Substrate Specificity
• ISSN: 0021-924X; 1756-2651
• DOI: 10.1093/oxfordjournals.jbchem.a132416

A 5′-nucleotidase which absolutely required magnesium ions was found in the cytoplasm of Bacillus sublilis K No. 321, an inosine-producing bacterium. Another cytoplasmic 5′-nucle- otidase of the bacterium which is activated only slightly by magnesium ions has been reported previously. The enzymes were completely separated from each other by Sephadex G-200 gel-filtration of the sonic extract of the cells. The magnesium-requiring enzyme had a molec ular weight of 23,000, an optimum pH of 7·5, and showed specific activation by Mg2+ The enzyme hydrolyzed the phosphomonoester bonds of both purine and pyrimidine nucleoside 5′-monophosphates, but did not hydrolyze UDP-glucose, 4-nitrophenylphosphate or bis(4 nitrophenyl)phosphate. The Km value for AMP was 0·25 mM. Mn2+ and Co2+ activated the enzyme only slightly in the absence of Mg2+ but were rather inhibitory in the presence of Mg2+ The enzyme was strongly inhibited by Zn2+ Cu2+ and Fe2+ The inorganic phosphate inhibition was of mixed type with respect to AMP, and the inhibitor constant for phosphate was 1·5 mM. From strain No. 231 a mutant was derived which contained only the cytoplasmic magnesium-requiring 5′-nucleotidase, and was deficient in other 5′-nucleotidases (the cell wall and membrane enzymes, the magnesium-non-requiring cytoplasmic enzyme, and the enzyme of culture supernatant). The mutant accumulated inosine in the culture medium to the same extent as the parent strain No. 231, indicating that the magnesium-requiring cytoplasmic enzyme is involved in the extracellular accumulation of inosine in B. subtilis K.